Evaluation of Commercial Anti-Listerial Products for Improvement of Food Safety in Ready-to-Eat Meat and Dairy Products

In ready-to-eat products, such as cooked ham, fresh cheese, and fuet in which Listeria monocytogenes is a concern, the use of biopreservation techniques represents an additional hurdle to inhibit pathogen growth during storage. The objective of this study was to apply several biopreservation techniques in three different food matrices to reduce the growth of Listeria innocua, used as a surrogate of L. monocytogenes. Several lactic acid bacteria, the bacteriocin nisin, the bacteriophage PhageGuard ListexTM P100, and the enzyme lysozyme were evaluated. Cooked ham treated with the bacteriophage PhageGuard ListexTM at 0.5% or with the lactic acid bacteria SafePro® B-SF-43 (25 g/100 kg) reduced L. innocua population to below the detection limit after 7 days of storage (4 °C plus modified atmosphere packaging). In fresh cheese, the application of PhageGuard ListexTM at 0.2 and 0.5% reduced L. innocua counts by more than 3.4 logarithmic units after 6 days at 4 °C. In fuet, the 1.0% of PhageGuard ListexTM reduced L. innocua population by 0.7 ± 0.2 logarithmic units in front of control with no significant differences to other evaluated biopreservative agents. The present results confirm that the application of biopreservation techniques was able to inhibit L. innocua in fuet, cooked ham, and fresh cheese, and suggest that the type of food matrix and its physicochemical characteristics influence the biopreservative efficacy.


Introduction
Listeria monocytogenes is a ubiquitous microorganism that can be found widely in the environment, but also in food processing plants where it can be protected from cleaning and disinfection operations due to its ability to produce biofilms. Therefore, L. monocytogenes can contaminate food at any point in its processing, from raw material production to household food handling. This microorganism causes invasive listeriosis which affects high-risk groups in the population: elderly people, newborns, immunocompromised people, and pregnant women.
Since L. monocytogenes is a quite resistant bacterium, able to grow at refrigeration temperatures (minimal growth temperature between −1.5 and 3.0 • C), at low pH and low water activity, ready-to-eat (RTE) foods with long shelf life are considered risky food products [1]. The main categories of RTE foods implicated in human listeriosis are 'meat and meat products', 'fish and fish products', and 'milk and milk products' [2]. Although the presence of L. monocytogenes in RTE foods is legislated in the European Union (EU) since 2006 as a food safety criterion (Commission Regulation (EC) 2073/2005), in the period from 2009-2013 a statistically significant increasing trend was observed in the EU [2]; meanwhile, the trend in the period from 2017-2021 was stable (neither increase nor decrease in the number of cases was reported) [3]. In 2021, listeriosis was the fifth most commonly reported zoonosis in humans in the EU [3]. It caused 2183 confirmed cases of invasive listeriosis with 923 hospitalisations and 196 deaths [3].
the Catalonia region (Spain) [26]. During its processing, the product undergoes fermentation that reduces its pH and water activity, increasing the stability of the product [27]. However, the presence and growth of L. monocytogenes in low-acidic fermented products has been reported [27][28][29].
In experimental studies conducted at pilot plant scale, L. monocytogenes cannot be used because of the risk of cross-contamination in the production of other foods. Therefore, a non-pathogen surrogate organism such as Listeria innocua must be used [30][31][32]. In addition, several authors have detected L. monocytogenes and L. innocua in the same samples from meat and dairy facilities [33][34][35]. Presence of L. innocua in food products is also a concern since it is a reservoir of resistance gens which may transfer between bacterial species. Gómez et al. [36] studied the resistance pattern of Listeria strains isolated from RTE meat products to various antibiotics. As a conclusion, they observed a multidrug resistance in 24 up to 71 Listeria isolates, with a higher prevalence of multidrug resistance in L. innocua specie (13.9%) than in L. monocytogenes specie (2.9%). In this scenario, control of both species, L. monocytogenes and L. innocua, could improve microbial food safety. This study aimed to assess the suitability of different commercial biopreservatives, including LAB, the bacteriocin nisin, the bacteriophage Listex TM P100, and the enzyme lysozyme to reduce the population of a Listeria innocua cocktail, used as an L. monocytogenes surrogate, in three different RTE foods (sliced cooked ham, fresh cheese, and fuet).

Effect of Antimicrobials on Cooked Ham
A first challenge test was carried out on cooked ham plugs at worst-case conditions (13 • C) to select the most effective techniques. A remarkable L. innocua population increase was observed in the control (inoculated only with L. innocua) from the initial counts (2.2 ± 0.5 log cfu/g) to 9.0 ± 0.2 log cfu/g after 6 days of storage at 13 • C. At the end of storage (6 days), in four out of seven of the evaluated treatments L. innocua population increased considerably in cooked ham plugs (between 5.8 and 6.6 logarithmic units), close to the increase observed in the control treatment (6.8 ± 0.2 logarithmic units). In contrast, L. innocua population increased only by 1.3 ± 0.3, 2.2 ± 0.7, and 3.9 ± 0.8 logarithmic units in the treatments with L. rhamnosus GG, SafePro ® B-SF-43, and PhageGuard Listex TM , respectively. Under the same storage conditions (13 • C), a second challenge test was carried out to elucidate if a reduced bacteriophage dose was also effective against L. innocua. The results showed that the three studied doses of PhageGuard Listex TM (1, 0.5, and 0.2%) had the same efficacy against L. innocua when applied to the surface of cooked ham plugs. Therefore, only the lower doses of bacteriophage were evaluated (0.2 and 0.5%) in the assay carried out at the recommended storage temperature (4 • C and air atmosphere).
During the evaluation of the selected biopreservative on cooked ham plugs stored under desirable refrigerated temperature (4 • C), only the application of the highest dose of PhageGuard Listex TM (0.5%) produced a significant reduction in L. innocua population after 3 days (Figure 1). In contrast, after 10 days of storage, all the treatments reduced L. innocua population and reached counts below 2.0 log cfu/g; meanwhile, in the control counts, it increased up to 3.4 ± 0.1 log cfu/g. The PhageGuard Listex TM at 0.5% reached the highest reduction (3.0 ± 0.2 logarithmic units) followed by the lowest bacteriophage dose (2.6 ± 0.6 logarithmic units), SafePro ® B-SF-43 (1.7 ± 0.0 logarithmic reductions), and L. rhamnosus GG (1.4 ± 0.0 logarithmic units).
When a semi-commercial evaluation was carried out on cooked ham slices under modified atmosphere packaging (MAP) at 4 • C, an immediate reduction was observed on L. innocua counts in the PhageGuard Listex TM treatments after inoculation (Figure 2a). The decrease was higher for cooked ham treated with the highest bacteriophage concentration (0.5%, L. innocua population not detected after enrichment) and in the PhageGuard Listex TM 0.2% treatment, in which L. innocua decreased below the detection limit (<4 cfu/g). In contrast, L. innocua counts in SafePro ® B-SF-43 (1.3 ± 0.1 log cfu/g) were not significantly different from the control. Nevertheless, seven days after treatment, SafePro ® B-SF-43 reduced L. innocua population to no detected bacteria after enrichment as both PhageGuard Listex TM treatments. After 14 days, in the SafePro ® B-SF-43 treatment only one of the three trays contained viable cell counts of L. innocua, which were below the detection limit (4 cfu/g); meanwhile, the PhageGuard Listex TM treatments (0.2 and 0.5%) reduced L. innocua to no detected bacteria after enrichment. When a semi-commercial evaluation was carried out on cooked ham slices modified atmosphere packaging (MAP) at 4 °C, an immediate reduction was obser L. innocua counts in the PhageGuard Listex TM treatments after inoculation (Figure 2 decrease was higher for cooked ham treated with the highest bacteriophage concen (0.5%, L. innocua population not detected after enrichment) and in the Phage Listex TM 0.2% treatment, in which L. innocua decreased below the detection limit (<4 In contrast, L. innocua counts in SafePro ® B-SF-43 (1.3 ± 0.1 log cfu/g) were not signif different from the control. Nevertheless, seven days after treatment, SafePro ® B-SF duced L. innocua population to no detected bacteria after enrichment as both Phage Listex TM treatments. After 14 days, in the SafePro ® B-SF-43 treatment only one of th trays contained viable cell counts of L. innocua, which were below the detection l cfu/g); meanwhile, the PhageGuard Listex TM treatments (0.2 and 0.5%) reduced L. i to no detected bacteria after enrichment.
Regarding the evolution of the biopreservation techniques in treated cooke stored at 4 °C under MAP, the counts of Leuconostoc carnosus (LAB formulated in Sa B-SF-43) increased from 5.7 ± 0.1 log cfu/g to 7.3 ± 0.1 log cfu/g during the first 7 d storage ( Figure 2b) and up to 8.9 ± 0.1 log cfu/g at the end of storage, while bacterio levels did not increase during storage compared with initial bacteriophage counts 0.2 and 8.0 ± 0.1 log pfu/g for 0.2% and 0.5% of PhageGuard Listex TM , respectively) 1 shows the O2 and CO2 gas concentration composition of trays measured befor pling. The CO2 concentration in trays treated with SafePro ® B-SF-43 increased from 3.4 to 36.6 ± 1.5% after 14 days of storage at 4 °C. Conversely, in the control (L. i only) and trays treated with PhageGuard Listex TM , the CO2 concentration decrease the storage time up to 13.3 ± 0.4% with no significant difference between these treat Values are means ± standard deviations of three biological replicates. Bars marked with different letters (a, b and c) represent statistically significant differences among treatments (p < 0.05) at each sampling time.  Regarding the evolution of the biopreservation techniques in treated cooked ham stored at 4 • C under MAP, the counts of Leuconostoc carnosus (LAB formulated in SafePro ® B-SF-43) increased from 5.7 ± 0.1 log cfu/g to 7.3 ± 0.1 log cfu/g during the first 7 days of storage ( Figure 2b) and up to 8.9 ± 0.1 log cfu/g at the end of storage, while bacteriophage levels did not increase during storage compared with initial bacteriophage counts (7.7 ± 0.2 and 8.0 ± 0.1 log pfu/g for 0.2% and 0.5% of PhageGuard Listex TM , respectively). Table 1 shows the O 2 and CO 2 gas concentration composition of trays measured before sampling. The CO 2 concentration in trays treated with SafePro ® B-SF-43 increased from 25.8 ± 3.4 to 36.6 ± 1.5% after 14 days of storage at 4 • C. Conversely, in the control (L. innocua only) and trays treated with PhageGuard Listex TM , the CO 2 concentration decreased with the storage time up to 13.3 ± 0.4% with no significant difference between these treatments.

Effect of Antimicrobials in Fresh Cheese
When the biopreservative agents' efficacy was evaluated in fresh cheese, antimicrobial agents were introduced directly into the milk used to elaborate the cheese. The effect of the biopreservative treatments applied during the elaboration of fresh cheese is shown in Figure 3. A remarkable L. innocua population increase was observed in the control treatment, from the initial population (3.0 ± 0.1 log cfu/g) to 5.3 ± 0.1 log cfu/g after 6 days of storage at 4 • C. Meanwhile, most of the evaluated lactic acid bacteria did not reduce L. innocua population, while L. rhamnosus GG obtained a slight reduction after 3 days (0.6 ± 0.2 logarithmic units) and 6 days (0.8 ± 0.1 logarithmic units) of storage in front of control. Conversely, all the evaluated doses of PhageGuard Listex TM reduced L. innocua population below the detection limit (<10 cfu/g) after inoculation. However, only 0.5 and 1% of PhageGuard Listex TM treatments prevented L. innocua regrowth after 6 days of storage.
Under semi-commercial conditions, L. innocua population increased in the control from 3.2 ± 0.1 log cfu/g to 5.8 ± 0.2 log cfu/g after 6 days in cheese covered with liquid and stored at 4 • C (Figure 4a). After the obtention of the manufactured cheese, a biopreservative effect against L. innocua was observed in all the evaluated treatments with reductions higher than 3 logarithmic units in both PhageGuard Listex TM treatments (0.2 and 0.5%) and 0.3 ± 0.1 logarithmic unit reductions in L. rhamnosus GG treatment. The decrease of L. innocua population in the 0.5% PhageGuard Listex TM treatment was remarkable because it reduced the pathogen population below the detection limit (<10 cfu/g) and no L. innocua was detected after enrichment throughout storage. After 6 days of storage, L. rhamnosus GG treatment reduced L. innocua population up to 4.5 ± 0.1 log cfu/g (1.4 ± 0.1 logarithmic units in front of control) and 0.2% PhageGuard Listex TM obtained reductions of 3.4 ± 0.4 logarithmic units. Under semi-commercial conditions, L. innocua population increased in the co from 3.2 ± 0.1 log cfu/g to 5.8 ± 0.2 log cfu/g after 6 days in cheese covered with liquid stored at 4 °C ( Figure 4a). After the obtention of the manufactured cheese, a biopres tive effect against L. innocua was observed in all the evaluated treatments with reduc higher than 3 logarithmic units in both PhageGuard Listex TM treatments (0.2 and 0 and 0.3 ± 0.1 logarithmic unit reductions in L. rhamnosus GG treatment. The decrease innocua population in the 0.5% PhageGuard Listex TM treatment was remarkable beca reduced the pathogen population below the detection limit (<10 cfu/g) and no L. inn was detected after enrichment throughout storage. After 6 days of storage, L. rham GG treatment reduced L. innocua population up to 4.5 ± 0.1 log cfu/g (1.4 ± 0.1 logarit units in front of control) and 0.2% PhageGuard Listex TM obtained reductions of 3.4 logarithmic units.  Under semi-commercial conditions, L. innocua population increased in the control from 3.2 ± 0.1 log cfu/g to 5.8 ± 0.2 log cfu/g after 6 days in cheese covered with liquid and stored at 4 °C ( Figure 4a). After the obtention of the manufactured cheese, a biopreservative effect against L. innocua was observed in all the evaluated treatments with reductions higher than 3 logarithmic units in both PhageGuard Listex TM treatments (0.2 and 0.5%) and 0.3 ± 0.1 logarithmic unit reductions in L. rhamnosus GG treatment. The decrease of L. innocua population in the 0.5% PhageGuard Listex TM treatment was remarkable because it reduced the pathogen population below the detection limit (<10 cfu/g) and no L. innocua was detected after enrichment throughout storage. In this assay, the bacteriophage P100 concentrations in the obtained fresh cheeses were 6.3 ± 0.1 log pfu/g and 6.7 ± 0.1 log pfu/g in 0.2 and 0.5% bacteriophage treatment, respectively (Figure 4b). Regardless of the initial dose of bacteriophage applied in the milk (7.2 ± 0.1 log pfu/mL and 8.0 ± 0.0 log pfu/mL in treatment with PhageGuard Listex TM 0.2% and 0.5%, respectively), the bacteriophage increased up to 7.3 log pfu/g in fresh cheese. In contrast, the initial population of L. rhamnosus GG in the obtained fresh cheese was 8.6 ± 0.3 log cfu/g and levels remained stable throughout storage.
During the manufacturing of fresh cheese from the semi-commercial assay, the population of L. innocua and the biopreservative agents was also determined in the recovered whey after curd separation. The population of L. innocua in the recovered whey varied among treatments. Meanwhile, in the control and L. rhamnosus GG treatment, counts of 2.0 ± 0.0 and 2.2 ± 0.0 log cfu/mL of L. innocua were recovered in the whey; in both treatments with PhageGuard Listex TM the counts of L. innocua were below the detection limit (<10 cfu/mL). In addition, all used biopreservants were recovered in the whey with populations of 7.5 ± 0.1 log CFU/mL in L. rhamnosus GG treatment, 7.2 ± 0.0 log pfu/mL for 0.5% PhageGuard Listex TM , and 6.5 ± 0.1 log pfu/mL for 0.2% PhageGuard Listex TM .
The yield of the obtained cheeses with each biopreservative agent was determined. From 400 mL of inoculated milk, in the control treatment 94.75 g of fresh cheese was obtained, 97.97 g in the L. rhamnosus GG treatment, 91.99 g in the PhageGuard Listex TM 0.5% treatment, and 95.25 g in the PhageGuard Listex TM 0.2% treatment.
The yield of the produced cheese ranged between 0.24 and 0.25 kg/L of milk.

Effect of Antimicrobials in Fuet
The efficacy of seven biopreservative agents to control L. innocua population in fuet after ripening and post-cold storage was evaluated. In the fuet control (meat inoculated only with L. innocua), the pathogen population increased from the initial concentration (4.5 ± 0.6 log cfu/g) to 5.3 ± 0.1 log cfu/g after fermentation (24 h at 22 • C), with an increase of 0.7 ± 0.1 logarithmic units. In contrast, during the ripening process (7 days at 15 • C and 75% R.H.), the population of L. innocua in the control treatment reduced by 0.9 ± 0.1 logarithmic units with a final count of 4.3 ± 0.1 log cfu/g ( Figure 5). However, the population increased (0.5 ± 0.4 logarithmic units) during post-cold storage, reaching a final population of 4.8 ± 0.4 log cfu/g. In treated samples, SafePro ® B-SF-43 obtained the highest population reduction (0.5 ± 0.3 logarithmic reductions) after the ripening step in front of the control. Lower L. innocua counts after cold storage were observed in the treatments SafePro ® B-SF-43 (3.8 ± 0.3 log cfu/g), SafePro ® B-LC-20 (3.9 ± 0.5 log cfu/g), and PhageGuard Listex TM (4.0 ± 0.3 log cfu/g). These three treatments reduced pathogen population around 1 logarithmic unit in front of control at the end of shelf life. The biopreservative treatments that showed the best results were evaluated against L. innocua in fuet ( Figure 6). In this evaluation, the population of L. innocu enumerated immediately after inoculation (before stuffing in pork casing), after 2 fermentation, and at the end of the ripening (Figure 6a). After inoculation, the popu of L. innocua in the control was 4.8 ± 0.5 log cfu/g, and a slight reduction (0.5 ± 0.2 log mic unit) was reached in PhageGuard Listex TM  The biopreservative treatments that showed the best results were evaluated again against L. innocua in fuet ( Figure 6). In this evaluation, the population of L. innocua was enumerated immediately after inoculation (before stuffing in pork casing), after 24 h of fermentation, and at the end of the ripening (Figure 6a). After inoculation, the population of L. innocua in the control was 4.8 ± 0.5 log cfu/g, and a slight reduction (0.5 ± 0.2 logarithmic unit) was reached in PhageGuard Listex TM treatment with an L. innocua population of 4.3 ± 0.2 log cfu/g. After 24 h of fermentation, no reduction was observed in treatment SafePro ® B-SF-43; meanwhile, significant reductions were reached in the treatments Phage-Guard Listex TM Table 2 shows the evolution of pH during fuet production. In all treatments, pH decreased more than one unit after fermentation (from pH 6.33 ± 0.02, sausage mixture before stuffing). The pH reduction observed in the L. rhamnosus GG treatment after fermentation reached the lowest pH value of 5.06 ± 0.04.   Table 2 shows the evolution of pH during fuet production. In all treatments, pH decreased more than one unit after fermentation (from pH 6.33 ± 0.02, sausage mixture before stuffing). The pH reduction observed in the L. rhamnosus GG treatment after fermentation reached the lowest pH value of 5.06 ± 0.04.

Discussion
This study aimed to compare the efficacy of several biopreservative agents to control a four-strain L. innocua cocktail on sliced cooked ham (the evaluated treatments were PhageGuard Listex TM  and NisinZ ® ). The most appropriate time to apply the biopreservative agents was selected for each food matrix considering the physicochemical characteristics of the finished food, the microbiota of the raw material, and the manufacturing process steps. Therefore, cooked ham was surface treated after the slicing operation because it could be get contaminated if food manufacturing practices are not applied correctly in the operations that take place after pasteurization or thermal treatment, the last operation aimed to reduce microorganisms.
In the case of fresh cheese, the biopreservative agents were added after milk treatment, since L. monocytogenes contamination in cheese could be due to survived bacteria to a weak thermal treatment or from cross-contamination after thermal treatment [25,37]. Finally, in fuet, the biopreservative agents were also added to the raw materials at the beginning of the production process because no step in the production process has the purpose of reducing microorganism concentration in the raw material. Nevertheless, pH reduction and the reduction in water activity occur in fuet during the ripening process could avoid L. monocytogenes multiplication. In addition, the introduction of biopreservative agents before a homogenization step allows the spreading of the agents uniformly in food.
In a previous study, Szczawinski et al. [38] demonstrated the ability of L. monocytogenes to grow on contaminated cooked ham at different temperatures (3,6,9,12, and 15 • C), increasing the growth rate with the temperature increase. This study highlights L. innocua's ability to grow on cooked ham plugs stored at 4 • C with an increase of 0.6 logarithmic units after 6 days of storage. However, the pathogen maintained constant population counts when the sliced cooked ham was packaged under modified atmosphere. Furthermore, the L. innocua population increased by 2. 6 logarithmic units in the fresh cheese after 6 days of storage, although it was packaged in a covering liquid with 2% sodium chloride and stored at 4 • C. In queso fresco, which has similar characteristics to fresh cheese, Lourenço et al. [39] observed an L. monocytogenes population increase from 3.5 log cfu/g to 7.0 log cfu/g after 11 days of storage at 4 • C, although they inoculated L. monocytogenes in the curd instead of the milk. In contrast, the ripening step of the production process of the fuet caused a decrease of 0.3 logarithmic units of L. innocua after 7 days at 15 • C at 75% humidity. Previously, Porto-Fett et al. [40] validated that L. monocytogenes population reduced approximately by 3.6 log cfu/g during the preparation and storage of fuet.
To our knowledge, no previous research has compared the efficacy of different biopreservative agents in three food matrices of RTE products with such different characteristics. In this study, on sliced cooked ham, the most effective anti-listerial treatment was PhageGuard Listex TM followed by the SafePro ® B-SF-43 agent. Furthermore, PhageGuard Listex TM showed the most effective anti-listerial activity against L. innocua in packaged fresh cheese followed by L. rhamnosus GG. In both matrices, the evaluated biopreservation techniques reduced L. innocua counts by more than 1.5 logarithmic units in front of the control. Conversely, the maximum reduction observed in the manufactured fuet was 0.5 logarithmic units with no significant difference among PhageGuard Listex TM , SafePro ® B-LC-20, SafePro ® B-SF-43, and L. rhamnosus GG.
The manufacturer's recommendation for SafePro ® B-SF-43 application was 25 g/ 100 kg of product, which corresponded to 5.7 ± 0.1 log/cfu of Leuc. carnosum per gram in our evaluated sliced cooked ham. Budde et al. [41] observed that the rate of inhibition of L. monocytogenes in a cooked ham depended on the initial concentration of Leuc. carnosum 4010 applied. They reported that when the initial concentration of the Leuc. carnosum was 6.3 × 10 6 cfu/g, L. monocytogenes inhibition was observed after 7 days. Meanwhile, when a lower initial concentration (1.2 × 10 5 cfu/g) was applied the inhibition effect was observed after 14 days. This may be explained by a higher initial lactic acid bacteria population that reached the exponential growth phase earlier. Nevertheless, regardless of the initial Leuc. carnosum concentration, at the end of storage (28 days at 5 • C) the cell counts of L. monocytogenes were reduced to below 10 cfu/g in both treatments. In this study, Leuc. carnosum reached the exponential growth phase after 7 days of storage at 4 • C in the sliced cooked ham. Bacteriocin production by LAB usually starts in this growth phase [42]. In our study, L. innocua inhibition was not observed immediately after the application of SafePro ® B-SF-43 on the treated slices of cooked ham, and it was delayed to 7 days, when bacteriocins could have been produced, similarly to Budde et al.'s [41] results. The same authors also described the strong anti-listerial activity of Leuc. carnosum without producing undesirable flavour in the evaluated RTE meat foods. Although Leuc. carnosum established in the fuet, it multiplied slightly, around 0.4 logarithmic units after 7 days of ripening. Meanwhile, on the cooked ham, the bacteria increased fourfold compared to the fuet (1.6 logarithmic units). The adverse environment (low pH and a w , and microbial competition with indigenous fermentation strains) in the fuet could have caused the lower Leuc. carnosum multiplication and, consequently, lower bacteriocin production than in the cooked ham, in which higher anti-listerial activity was observed. The same anti-listerial rate activity was observed when the SafePro ® B-LC-20 was applied in the contaminated mixed meat used to elaborate the fuet. Pediococcus acidilactici is the bacteria lyophilizate in SafePro ® B-LC-20 formulation. Nieto-Lozano et al. [43] evaluated the inhibitory effect of P. acidilactici MCH14 against L. monocytogenes in Spanish dry-fermented sausage. This pediocin-producing strain reduced the L. monocytogenes counts by 2 logarithmic units compared to the control after 30 days of storage. They inoculated the minced meat at a P. acidilactici initial concentration of 5 × 10 6 cfu/g. Although higher initial concentration was applied in our fuet (7.7 ± 0.1 log cfu/g), a lower anti-listerial activity was observed. This could be due to the temperature used in our experiment, since a previous study noticed that the activity of P. acidilactici was more effective at 4 • C than 15 • C in raw meat [44]. P. acidilactici is used as a starter culture for its capacity to reduce food pH; its efficacy as an antimicrobial against Listeria has also been confirmed. In addition, Komora et al. [45] and Barbosa et al. [46] concluded that P. acidilactici should be considered a potentially useful probiotic.
Dairy products have extensively been used as food vehicles for L. rhamnosus GG, which is the most introduced probiotic strain in food. Rubio et al. [47] confirmed its suitability as a starter culture in cured sausage, and in this study we have evaluated its suitability as an anti-listerial agent in three treated food matrices. The highest L. innocua reduction was obtained in the fresh cheese with 1.4 logarithmic units in front of control, as in the cooked ham plugs stored under air conditions a 4 • C, whilst in fuet a decrease of 0.5 logarithmic units was achieved at the end of the ripening step. Although the probiotic strain was more effective in the fresh cheese than in the fuet, L. rhamnosus GG maintained the population counts above 10 8 cfu/g until the end of storage in both matrices. Therefore, the obtained fresh cheese and fuet in this study could be commercialised as functional foods while L. rhamnosus GG improves food safety against L. monocytogenes.
Generally, the PhageGuard Listex TM was the most effective biopreservative agent among the evaluated techniques because it showed effective anti-listerial activity in the three evaluated food matrices. In addition, a large reduction of L. innocua population was observed in the fresh cheese and the cooked ham. Several authors have also observed anti-listerial activity when applying the bacteriophage P100 in these two food matrices [21,48,49]. Similarly to our results, Holck et al. [21] obtained a 1 logarithmic reduction of L. monocytogenes on cooked ham after application of the bacteriophage P100 (5 × 10 7 pfu/cm 2 ) and the surviving pathogen population remained constant until 7 days of storage at 4 • C. Furthermore, higher pathogen reduction after inoculation was observed when we increased the inoculation levels of Phage-Guard Listex TM on the sliced cooked ham. The same behaviour tendency was observed in the fresh cheese. Carlton et al. [50] also noticed that the effect of bacteriophages varies with the type of product and is strongly dose dependent. This could be explained by the fact that a high concentration of bacteriophage per unit area is required to ensure interaction with the target pathogen on food surfaces [48]. The lowest initial dose of PhageGuard Listex TM allowed L. innocua regrowth in the fresh cheese throughout storage. Regarding bacteriophage multiplication ability, we observed that in the fresh cheese bacteriophage concentration increased with time. The self-replication ability of bacteriophage only occurs if there is still a host present in the food matrix, and the observed biopreservative population increase was in accordance with L. innocua survival after the treatment application of PhageGuard Listex TM (0.2%). Therefore, on cooked ham, the P100 population remained stable from inoculation to the end of the evaluation because no L. innocua survived after inoculation. Conversely to the lower reduction levels obtained in the fuet, Gutiérrez et al. [51] and Komora et al. [45] observed a reduction of 3 logarithmic units of L. monocytogenes counts respectively in a Spanish dry-cured ham and in a fermented meat sausage (Alheira). However, the addition of extra hurdles, such as high pressure, were necessary to reduce L. monocytogenes to below detection limits after 60 days of storage in the fermented sausage. Although an increase of the bacteriophage P100 population was noticed in the fresh cheese when L. innocua cells were present, in the fuet the bacteriophage population did not increase. Nevertheless, a large quantity of L. innocua was present in all evaluated sampling times in the fuet. This could be due to the food's characteristics, such as low moisture and low pH, which did not allow bacteriophage multiplication. In addition, several authors described that in nonliquid food matrices the phage particles appear to become immobilized soon after addition due to limited interaction between the phage and the target bacteria [50,52]. For instance, when Listex ® P100 was applied on fresh-cut melon and in melon juice, Oliveira et al. [53] observed the highest effectivity in the liquid matrix.
Although the results obtained in this study indicate the feasibility of applying biopreservation in the selected food matrices, it is important to highlight this study's limitations. Firstly, due to the impossibility to work with the human pathogenic L. monocytogenes, L. innocua was used as a surrogate. However, previously, an in vitro assay was conducted to determine the sensitivity of L. innocua selected cocktail in comparison to a cocktail of five strains of L. monocytogenes against the different selected biopreservative agents. The results showed that both Listeria cocktails presented similar sensibility. In addition, when slight differences in sensibility were observed, L. monocytogenes cocktail was more sensitive than the evaluated L. innocua cocktail. For this reason, we determined that the selected L. innocua strains cocktail was adequate. Secondly, the objective of this study was to evaluate the effect of several biopreservative agents against L. innocua in three food matrices susceptible to be linked to listeriosis. Therefore, to quantify L. innocua population reduction, the initial levels of L. innocua in raw ingredients was above 100 cfu/g, which is a relatively high population. Thirdly, we used a small sample size in this study. However, the aim was to evaluate several options of commercial biopreservatives to obtain knowledge of which could be the best option to food producers. Regardless of the obtained results and the background information, before the application of a new biocontrol tool in a production system, its effectiveness must be evaluated in the specific product considering the food composition, the food physicochemical characteristic, such as pH and water availability, and the production process. Likewise, the evaluated sampling times were approximations of reality, and before biopreservative application in the food chain, longer storage times should be evaluated.

Products
The biopreservatives' efficacy was evaluated in three different food matrices: cooked ham, fresh cheese, and dry-cured Spanish pork sausage (fuet). A commercial cooked ham was used to evaluate the anti-listerial efficacy of the biopreservatives.
The fuet and the fresh cheese were elaborated in the laboratory from the raw materials, pork minced meat and cow pasteurized milk, purchased from a supermarket. Both manufactured foods did not receive any thermal treatment.

Strains, Commercial Biopreservatives, and Stock Culture Preparation
A cocktail of four strains of L. innocua was used in this study as a surrogate of L. monocytogenes. These strains were: L. innocua CECT 910, L. innocua CECT 4030 and L. innocua CECT 8848 from Colección Española de Cultivos Tipo (CECT), and L. innocua TA-1.17 from the Department of Food Technology (UdL) collection. The stock cultures were streaked onto TSA (Tryptone Soy Agar; Biokar, Allone, France) supplemented with yeast extract (6 g/L, Biokar, France; Tryptone Yeast Extract Soy Agar, TYESA) and incubated at 37 • C for 24 h. For each strain, a single colony was picked from the plate and transferred to 10 mL of TSB (Tryptone Soy Broth; Biokar, France) supplemented with yeast extract (6 g/L, Tryptone Yeast Extract Soy Broth, TYESB) and incubated at 37 • C for 20-22 h. The four strains of L. innocua were mixed in equal amounts regarding cell numbers to obtain the same concentration of each strain (10 8 cfu/mL). The cocktail was centrifuged (Sorvall Legend XTR, Thermo Scientific, Waltham, MA, USA) for 10 min at 8900× g, and the pellet was resuspended in saline solution (8.5 g/L NaCl, Thermo Fisher, Markham, Canada). For food inoculation, a serial dilution of the L. innocua cocktail was prepared in saline peptone solution (SP, 8.5 g/L NaCl and 1 g/L peptone (Biokar, France)) to obtain the desired concentration.
The anti-listerial agents used were the bacteriophage PhageGuard Listex TM (Micreos, Wageningen, the Netherlands), the formulated cultures SafePro ® B-LC-20, SafePro ® B-LC-48, and SafePro ® B-SF-43 (CHR-Hansen, Horsholm, Denmark), and Fermitrat-Export ® and Fermitrat-S3 ® (Amerex, Colmenar Viejo, Spain), the probiotic strain L. rhamnosus GG (ATCC 53103), the bacteriocin NisinZ ® (Handary, Evere, Belgium), and the enzyme Lysoch ® L4 (Handary, Evere, Belgium). The evaluated biopreservative agents are currently commercialised products in the EU. According to legislation and manufacturers' instructions, each biopreservative agent was evaluated in the food matrix in which it is recommended. To apply all biopreservation techniques using the same method, the lyophilised or dehydrated products were suspended in sterile water prior their application. When necessary, the biopreservative agent's suspension was serially diluted to treat the samples with the same volume of each anti-listerial agent.
Prior to the evaluation of the most suitable techniques on slices of cooked ham in a commercial scenario (4 • C and MAP), two experiments were performed in cooked ham portions under air conditions at different temperatures (13 • C and 4 • C). For these experiments, a slice of cooked ham was cut into plugs of 1.5 cm diameter and 0.5 cm thick (approximately 1 g/plug). Then, 10 µL of the L. innocua cocktail was spread over the plug surface to obtain a concentration of 100 cfu/g. After 15 min at room temperature, of calcium chloride and 0.7 mL/L of rennet were added to each treatment and milk was incubated at 40 • C for 30 min. Each curd was cut into 1 cm cubes, stirred, and maintained at 40 • C for 30 min. Whey was drained off using a cotton cheesecloth and small half spheres of cheese (2.6 cm diameter) were made using a food grade silicone mould. Nine cheeses (approximately 4.5 g/unit) were obtained from each treatment. Prior to packaging, cheeses were dipped in a salt slurry of 20% sodium chloride for 15 min. Three cheeses of each treatment were introduced to a plastic bag (polyethylene, 14 × 9 cm) with 15.0 mL of covering liquid (containing 2% NaCl) and thermal sealed. Packaged samples were stored at 4 • C for 6 days. L. innocua and biopreservative agents' population were enumerated at initial time and after 3 and 6 days of storage as described below (4.4.). In the semi-commercial assay, the resulting fresh cheeses were weighed, and the yield of each treatment was determined.

Dry-Cured Spanish Pork Sausage (Fuet)
For fuet, the sausage was prepared with minced pork meat (70:30, lean:fat ratio) and a commercial preparation (Teifel Longaniza 807, marca Teixidor S.L., Spain) was added (52 g/kg). In the first trial, 3.0 kg of sausage mixture was inoculated with 1 mL/100 g of L. innocua cocktail to obtain a population of 10 5 cfu/g. After homogenization, inoculated sausage mixture was divided into nine batches of 300 g. Eight treatments and an L. innocua control were evaluated: (1) L. innocua control (no treatment), (2) SafePro ® B-LC-20 (25 g/100 kg), (3) SafePro ® B-LC-48 (12.5 g/100 kg), (4) SafePro ® B-SF-43 (25 g/100 kg), (5) L. rhamnosus GG (10 8 cfu/g), (6) Fermitrat-S3 ® (10 g/100 kg), (7) Fermitrat-Export ® (150 g/100 kg), (8) NisinZ ® (1 g/100 kg), and (9) PhageGuard Listex TM (1%). In the L. innocua control treatment, 1 mL/100 g of sterile water was spread into the inoculated sausage mixture. The same volume (1 mL/100 g) of each biopreservative agent suspension was spread individually and mixed. Each of the treated mixtures were stuffed into natural pork casings (30/32 diameter, packed in salt). Previously, pork casings were conditioned in tap water during 30 min and cleaned. Manufactured fuets (approximately 30 g/fuet) were placed in a fermentation chamber at 22 • C (saturated humidity) for 24 h to promote fermentation and pH decrease. Ripening of fuets took place at 15 • C and 75% relative humidity for 7 days. Prior to biopreservation assays, a trial was realised to determine the time required to achieve a half reduction of the initial weight of the fuets. After ripening, the inoculated and treated fuets were stored at 4 • C for 7 days. L. innocua population was enumerated after ripening and cold storage (7 days at 4 • C). After no observed differences among biopreservative and control treatments in the post-refrigerated storage time of the fuet, in the second assay we decided to focus on the production steps and evaluated the action of biopreservative agents only during processing. After the initial results, the most effective techniques were evaluated again at different steps of the fuet manufacture process. Treatments were: (1) L. innocua control (no treatment), (2) PhageGuard Listex TM (1%), (3) SafePro ® B-LC-20 (25 g/100 kg), (4) SafePro ® B-SF-43 (25 g/100 kg), and (5) L. rhamnosus GG (10 8 cfu/g). As previously described, 2.0 kg of sausage mixture was inoculated to obtain an initial population of 10 5 cfu/g of L. innocua cocktail. Then, the inoculated sausage mixture was divided into five batches (400 g) and each batch was treated individually with 1 mL/100 g of biopreservation suspension. The control was treated with sterile water. All fuets were subjected to a fermentation step (24 h) followed by a ripening of 7 days. L. innocua counts were determined after inoculation, after fermentation, and at the end of ripening, and biopreservative agents' populations were enumerated after their application and at the end of ripening as described below (4.4.), when weight, pH, and water activity (a w ) were also determined. The pH measurement was made using a handheld pH-meter (Testo 205, Testo SE & Co. KGaA, Titisee-Neustadt, Germany) and the AquaLab (Series 3 TE, Decagon Devices, Inc., Pullman, WA, USA) was used to determine the a w .

Food Sampling
To determine L. innocua population of the treated cylinders of cooked ham, a plug (approximately 1 g/plug) was placed in a sterile filter plastic bag (80 mL, BagPage ® Internscience BagSystem, Saint Nom, France) and diluted with 5 mL of buffered peptone water (BPW, Biokar, Allone, France). Samples were homogenized in a blender for 90 s and L. innocua population was enumerated as described below (4.4.2). Each treatment was evaluated in triplicate.
To enumerate pathogen and biopreservative agents' populations on sliced cooked ham during commercial assay, 3 trays were analysed for treatment at each sampling time. From each tray, 25 g of treated slices in a sterile filter bag (400 mL, BagPage ® Internscience BagSystem, France) was homogenised with 75 mL of half Fraser broth (Biokar, France) in a paddle blender for 90 s. One millilitre of the mixture was spread onto PALCAM agar (Biokar, France) by duplicate and incubated at 37 • C for 48 h. Then, the mixture was supplemented with the half Fraser supplement (Biokar, France) and incubated at 30 • C for 24 h. After that, the enriched sample was streaked onto PALCAM, and 0.1 mL was mixed with 10 mL of complete Fraser broth to allow the detection of L. innocua. In case of a negative result, enriched tubs were incubated at 30 • C for 24 h and streaked onto PALCAM and COMPASS agar (Biokar, France).
To enumerate the L.
innocua population in the fresh cheese and in the fuet, one gram of sample was placed in a sterile filter plastic bag (80 mL, BagPage ® Internscience BagSystem, France) and diluted with 9 mL of BPW. Samples were homogenized in a blender for 90 s and L. innocua population was enumerated as described below (Section 4.4.2). Each treatment was evaluated in triplicate. After enumeration, the mixture bags were incubated at 30 • C for 24 h. Then, 0.1 mL of incubated mixture was added to 9 mL of complete Fraser broth when enumeration was below the detection limit. Enriched tubs were incubated, streaked onto PALCAM agar, and plates were incubated at 37 • C for 24-48 h.

L. innocua Enumeration
For L. innocua enumeration, regardless of the matrices evaluated, the mixture (food homogenized in BPW) was serially diluted in SP, plated onto PALCAM agar, and incubated at 37 • C for 48 h. Plates were counted and results were expressed as log cfu/g.

Biopreservative Agents' Enumeration
To determine the biopreservative agent counts, the same samples obtained to enumerate L. innocua population were used. To enumerate LAB population (treatments: SafePro ® B-LC-20 (in fuet), SafePro ® B-SF-43 (in fuet and cooked ham), and L. rhamnosus GG (in fuet and fresh cheese)), the mixture dilution was plated onto de Man Rogosa and Sharpe agar (MRS, Biokar, France) and incubated at the LAB optimum temperature/time (SafePro ® B-SF-43 (Leuc. carnosum) at 25 • C for 5 days, and SafePro ® B-LC-20 (P. acidilactici) and L. rhamnosus GG at 37 • C for 24 h). Plates were counted and results were expressed as log cfu/g. Bacteriophage (P100) counts were determined using the soft agar overlay method following instructions given by the product supplier. Aliquots of 100 µL of the diluted food mixture were mixed with 50 µL of log-phase L. innocua (strain 2627 from Micreos collection) in 4.0 mL of Luria-Bertani agar (LB, 10 g/L of Pectic Digest of Meat USP (Biokar, France), 5 g/L of yeast extract, 10 g/L of NaCl, and 4 g/L agar (Condalab, Spain)). Instantly, the soft agar mixture was poured onto solid Brain Heart Infusion agar (BHI, Biokar, France) plates and plates were incubated for 24 h at 30 • C. After incubation, the visible plaques were counted and expressed as log particle forming unit (pfu)/g.

Statistical Analysis
The recovered L. innocua counts were transformed into log 10 to normalize the data. When pathogen detection was positive with an enumeration below the limit of detection (LOD), the count was considered a half of the LOD. In contrast, a negative result after enrichment was considered an absence of L. innocua. An analysis of variance (ANOVA) was performed to evaluate the significant effect of each biopreservative treatment at each evaluated time. In the biopreservative agents' enumeration, counts were transformed into log 10 to normalize the data. An analysis of variance (ANOVA) was performed to evaluate the significant effect of time at each evaluated treatment. Multiple comparisons were evaluated by Tukey's Honest Significant Difference (HSD) test with p < 0.05 using the JMP Pro 16 software (SAS Institute Inc., Cary, NC, USA).

Conclusions
The obtained results have shown that some currently commercialized biopreservation techniques have great potential in reducing the microbiological risk associated with the presence of L. monocytogenes in RTE foods. However, all biopreservative agents with the potential to be applied in a food product should be evaluated for each food matrix [49] to select the most efficient method for each product (higher effectivity with lower dose) and to identify the best production step for their introduction. In this sense, it is necessary to consider that the applied LAB cultures do not allow a thermal treatment whilst bacteriophage allows low thermal treatments. Therefore, use of the appropriate biopreservative agent in each RTE product could improve food safety for consumers. However, biopreservation should be used as an extra hurdle against L. monocytogenes in combination with the application of current good hygienic practices and good manufacturing practices.